The interior of San Isidoro surprises visitors with its bright light, the result of the abundance of large windows in the central nave and the south transept. The walls, both straight and curved, are decorated with checkered imposts, a motif that repeats uniformly throughout the space, while the capitals stand out for their richness and number, offering a varied and detailed sculptural repertoire.

The Main Chapel, built in the 16th century and attributed to Juan de Badajoz the Elder, was commissioned by Father Juan de Cusanza in 1513 to replace the old Romanesque chapel of the Infanta Urraca. In 1971, cleaning and restoration work was carried out on the floor, revealing the foundations of the Romanesque floor plan discovered by the architect Torbado, marked by a drawing on the pavement. The chapel is covered by a ribbed vault with tercelets, reflecting the transition between the late Gothic and the Renaissance tastes of the period.

The main altarpiece, of Gothic design, comes from the parish church of Pozuelo de la Orden (Valladolid) and was moved to San Isidoro in 1920. Carved between 1525 and 1530, it involved the participation of a master named Giralte, possibly Giralte of Brussels, and consists of twenty-four painted panels attributed by Chandler R. Post to a Master of Pozuelo, later identified as Lorenzo de Ávila, a follower of Juan de Borgoña, along with Antonio Vázquez and Andrés de Melgar, who were linked to the circle of Alonso Berruguete. The monstrance, a silverwork by M. García Crespo, holds the consecrated host, which is displayed day and night by papal privilege, shared with the Cathedral of Lugo; beneath it lies the neoclassical urn containing the remains of Saint Isidore, crafted in 1847 by the León silversmith Antonio Rebollo.

The Chapel of the Trinity, or of Saint Martino, was built around 1191 by the Isidorian canon Saint Martino behind the north apse, with the purpose of housing relics and serving as a common burial ground for the canons. Originally small, with a rectangular floor plan and a semicircular apse, and built with humble materials, it was transformed in the 16th century in the Hispano-Flemish style and renamed the Chapel of Santo Martino. The 17th-century altarpiece, the work of the woodcarver and cabinetmaker Pedro Margotedo, houses the image of the patron saint and the urn containing the saint’s remains.

The high choir, located at the foot of the church, dates from the first half of the 15th century and is in the Gothic style. It was built during the abbacy of Simón Álvarez, whose coat of arms can be seen on one of the keystones, thus blending into the temple’s rich and varied decoration.